Host–Guest Allosteric Control of an Artificial Phosphatase

The activity of many enzymes is regulated by associative processes. To model this mechanism, we report here that the conformation of an unstructured bimetallic Zn(II) complex can be controlled by its inclusion in the cavity of a γ-cyclodextrin. This results in the formation of a catalytic bimetallic site for the hydrolytic cleavage of the RNA model substrate HPNP, whose reactivity is 30-fold larger with respect to the unstructured complex. Competitive inhibition with 1-adamantanecarboxylate displaces the metal complex from the cyclodextrin decreasing the reactivity.


Experimental Procedures.
General: Solvents were purified by standard methods. All commercially available reagents and substrates were used as received. Water for kinetic experiments was purified with a Millipore MilliQ ® apparatus (18 M cm resitivity).
NMR spectra were recorded using a Bruker AV III 500 spectrometer operating at 500 MHz for 1 H, 125.8 MHz for 13 C. Chemical shifts are reported relative to internal Me 4 Si. Multiplicity is given as follow: s = singlet, d = doublet, t = triplet, q = quartet, qn = quintet, m = multiplet, br = broad peak.
ESI-MS mass spectra were obtained with an Agilent Technologies LC/MSD Trap SL mass spectrometer.
Stock solution of ligand 1, cyclodextrins and HPNP were prepared in water. pH of the ligand's stock solution was adjusted to 6.5 with 1 M NaOH. Stock solution of acid 1-adamantanecarboxilic was prepared in EPPS buffer at pH 8.0.
Kinetic experiments were performed with Cary 50 spectrophotometer equipped with thermostatic multiple cell holders. HPNP conversion was monitored by the increase of the absorption at 400 nm of the p-nitrophenolate. Kinetic analysis was performed with the initial rates methods. Absorbance data relative to the first part of the reaction, corresponding to a substrate conversion smaller than 10-15% were collected. In these conditions, substrate concentrations can be assumed to be constant and equal to the initial concentrations. Initial rates of each run wre calculated by a linear fitting of the data collected.
The slope values obtained from the fits were divided by the molar absorptivity () of p-nitrophenol S3 measured in the experimental conditions (1.65×10 5 cm -1 M -1 ) .. Experiments were performed in triplicate and data reported in the paper figures are the corresponding averages with standard deviations, which usually ranged between 5 and 10%.  Linear fits and the fitting of the pH vs rate profile was performed with the OriginPro 2018 software package. The equation used to fit the pH profile was: where K a is the apparent pK a of the deprotonable species.
The fitting of the other kinetic data was performed using the DynaFit software package. The scripts used are reported here (0.nnn is the initial extimation of the parameter to be adjusted in the fitting).
For the experiments revealing binding equilibria (Fig. 1B